Neo-tryptophan

ABSTRACT

The invention provides a novel amino acid, neo-tryptophan, as well as polypeptides containing this novel amino acid such as neurotensin analogs. In addition, the invention provides neo-tryptophan derivatives, serotonin-like neo-tryptophan derivatives, and polypeptides containing such derivatives. The invention also provides methods for making neo-tryptophan, neo-tryptophan derivatives, serotonin-like neo-tryptophan derivatives, and compositions containing these compounds. Further, the invention provides methods for inducing a neurotensin response in a mammal as well as methods for treating a mammal having a serotonin recognition molecule.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from the following U.S. ProvisionalApplications, U.S. Serial No. 60/081,356, filed Apr. 10, 1998, U.S.Serial No. 60/092,195, filed Jul. 9, 1998, U.S. Serial No. 60/098,119,filed Aug. 27, 1998 and U.S. Serial No. 60/112,137, filed Dec. 14, 1998.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Funding for the work described herein was provided by the federalgovernment, which may have certain rights in the invention.

BACKGROUND

1. Technical Field

The invention relates to a novel amino acid, neo-tryptophan, as well aspolypeptides containing this novel amino acid.

2. Background Information

Tryptophan is an essential component in human nutrition since it is notsynthesized by the body. In addition, tryptophan is a hydrophobic aminoacid that is part of many polypeptides.

Polypeptides as well as many other types of compounds such asneurotransmitters and drugs can exert profound effects on the body. Forexample, neurotensin (NT) is a tridecapeptide that inducesantinociception and hypothermia upon direct administration to brain.Systemic administration of NT does not induce these effects since NT israpidly degraded by proteases and has poor blood brain barrierpermeability. Currently, two NT receptors have been identified andcloned. The first NT receptor is designated NTR1, while the second isdesignated NTR2. Both NTR1 and NTR2 are G-protein coupled receptors thatare expressed by various brain tissues.

Serotonin (5HT) is a neurotransmitter that is essential to brainfunction. Multiple serotonin receptors and transporters have beenidentified and cloned. Briefly, de novo synthesis of serotonin fromtryptophan occurs in the cytoplasm of a cell. Once synthesized,vesicular monoamine transporters package the transmitter into vesicularcompartments so that its release can be regulated. Once released intothe synapse upon proper stimulation, the transmitter can bind specificserotonin receptors, can be degraded by specific enzymes, and/or can betransported back into a cell by specific plasma membrane serotonintransporters and then re-packaged into vesicles. Thus, both serotoninreceptors and transporters specifically recognize serotonin.

Apomorphine is an example of a drug that also influences brain function.Specifically, apomorphine is a non-selective dopamine D₂/D₃ receptoragonist. At low doses, apomorphine (e.g., 25-200 μg/kg) activatespre-synaptic receptors, while at higher doses (e.g., 600 μg/kg) itinfluences post-synaptic sites. Thus, the behavioral affects ofapomorphine vary with dosage. In mice and rats, high doses ofapomorphine cause a characteristic climbing behavior as well asoro-facial stereotypies such as sniffing and licking behaviors. Usingthese high doses of apomorphine, atypical neuroleptic compounds havebeen identified based on their ability to block potently the climbingbehavior while causing little change to the sniffing and lickingbehaviors. Both typical and atypical neuroleptic compounds have beenused to treat schizophrenia and other psychotic disorders. Atypicaldrugs are preferred because of their lower propensity to cause motorside effects (e.g., extrapyramidal side effects such as parkinsonism andtardive dyskinesia).

SUMMARY

The invention provides a novel amino acid, neo-tryptophan, as well aspolypeptides containing neo-tryptophan. In addition, the inventionprovides neo-tryptophan derivatives and serotonin-like neo-tryptophanderivatives as well as compositions containing these derivatives.Specifically, the invention provides neurotensin (NT) polypeptideanalogs as well as other polypeptides that contain neo-tryptophan. Theinvention also provides methods for making neo-tryptophan,neo-tryptophan derivatives, serotonin-like neo-tryptophan derivatives,and compositions containing such compounds. Further, the inventionprovides methods for inducing a neurotensin response in a mammal as wellas methods for treating a mammal having a serotonin recognitionmolecule.

One aspect of the invention features a polypeptide containingneo-tryptophan. The polypeptide can be substantially pure, andneo-tryptophan can be L-neo-tryptophan or D-neo-tryptophan. Thepolypeptide can interact with a neurotensin receptor, and can be aneurotensin analog with neo-tryptophan being located at amino acidposition 11 of neurotensin. The polypeptide can be NT64D, NT64L, NT65L,NT66D, NT66L, NT67L, NT69L, NT69L′, NT71, NT72, NT73, NT74, NT75, NT76,NT77, Ang1, Brdy1, or Lenk1.

In another aspect, the invention features an amino acid that isneo-tryptophan. The amino acid can be substantially pure, and can beL-neo-tryptophan or D-neo-tryptophan.

Another aspect of the invention features a neo-tryptophan derivative.The neo-tryptophan derivative can contain neo-tryptophan and a blockinggroup (e.g., Fmoc or Boc).

Another aspect of the invention features a serotonin-like neo-tryptophanderivative having the following structure:

with R₁, R₂, and R₃ being H, OH, CH₃, SH, F, NH₂, or COOH, and A beingzero, one, two, or three. For example, R₁ and R₃ each can be a hydroxylgroup, R₂ can be an amino group, and A can be zero.

Another aspect of the invention features a method of synthesizingneo-tryptophan. The method includes providing 4-hydroxymethyl indole,and substituting the hydroxyl group of the 4-hydroxymethyl indole with aglycyl unit to produce neo-tryptophan. The N-1 nitrogen of the4-hydroxymethyl indole can be protected by a protecting group (e.g.,Boc) that can be removed after the substitution. The method can include(a) providing 2-methyl-3-nitrobenzoic acid, (b) esterifying the2-methyl-3-nitrobenzoic acid to form an esterification product, (c)reacting the esterification product with N,N-dimethylformamidedimethylacetal to produce an enamine product, (d) performing reductivecyclization on the enamine product to produce a 4-substituted indolemethyl ester, (e) protecting the indole nitrogen of the 4-substitutedindole methyl ester with a Boc group, (f) reducing the protected4-substituted indole methyl ester with DIBAL to produceN-Boc-4-hydroxymethyl indole, (g) converting the N-Boc-4-hydroxymethylindole into benzylic bromide, (h) performing SN₂ displacement of thebromide of the benzylic bromide with a carbanion to producediastereomeric bislactim products, (i) isolating one of thediastereomeric bislactim products, (j) hydrolyzing the isolateddiastereomeric bislactim product to produce an aminoester product, (k)saponifying the aminoester product to produce an N^(ind)-t-Boc aminoacid, and (1) removing the Boc group to produce neo-tryptophan.

Another aspect of the invention features a method of synthesizing aneo-tryptophan derivative. The method includes providing 4-hydroxymethylindole having the N-1 nitrogen of protected by a protecting group, andsubstituting the hydroxyl group of the 4-hydroxymethyl indole with aglycyl unit to produce a neo-tryptophan derivative. The protecting groupcan include Boc. The method can include adding, after the substitution,an additional protecting group to the nitrogen within the glycyl unit.The additional protecting group can include Fmoc.

Another aspect of the invention features a method of making apolypeptide containing neo-tryptophan. The method includes providing aneo-tryptophan derivative, and linking an amino acid residue to theneo-tryptophan derivative to form the polypeptide containingneo-tryptophan (e.g., L-neo-tryptophan or D-neo-tryptophan). Theneo-tryptophan derivative can contains a blocking group attached to anitrogen atom.

Another aspect of the invention features a method of inducing aneurotensin response in a mammal (e.g., human). The method includesadministering an effective dose of a polypeptide containingneo-tryptophan to the mammal. The administration can be extracranial(e.g., intraperitoneal, intravenous, intradermal, subcutaneous, oral, ornasal). The neurotensin response can include antinociception,hypothermia, reduction in appetite, reduction in body weight, reductionin body weight gain, preventing or reducing catalepsy (e.g.,haloperidol-induced catalepsy), and/or reducing an effect of a CNSstimulant such as apomorphine, amphetamine, or cocaine. For example, theneurotensin response can include reducing a climbing behavior induced byapomorphine. The neurotensin response car include an antipsychoticeffect. For example, the polypeptide can reduce the signs or symptoms ofschizophrenia in the mammal. The polypeptide can interact with aneurotensin receptor (e.g., a rat or human neurotensin receptor). Thepolypeptide can be NT64D, NT64L, NT65L, NT66D, NT66L, NT67L, NT69L,NT69L′, NT71, NT72, NT73, NT74, NT75, NT76, or NT77.

Another embodiment of the invention features a method of treating amammal (e.g., human) having a serotonin recognition molecule. The methodincludes administering a composition to the mammal such that compositioninteracts with the serotonin recognition molecule (e.g., a serotoninreceptor such as a 5HT_(2A) receptor). The composition includesneo-tryptophan, a neo-tryptophan derivative, or a serotonin-likeneo-tryptophan derivative. The composition can include a polypeptide.

Another aspect of the invention features a method for screening apolypeptide for in vivo use. The method includes contacting apolypeptide containing neo-tryptophan with a protease, and determiningwhether or not the polypeptide remains intact.

Another aspect of the invention features the use of a polypeptidecontaining neo-tryptophan in the manufacture of a medicament fortreating a mammal.

In another embodiment, the invention features the use of a compound inthe manufacture of a medicament for treating a mammal. The compoundcontains neo-tryptophan, a neo-tryptophan derivative, or aserotonin-like neo-tryptophan derivative.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram depicting the chemical structure of tryptophan,iso-tryptophan, neo-tryptophan, and tyrosine.

FIG. 2 is a diagram depicting the chemical structure of serotonin andserotonin-like neo-tryptophan derivatives. X can be any chemical groupor modification.

FIG. 3 is a diagram depicting a scheme used to chemically synthesizeboth D- and L-neo-tryptophan. Series “a” compounds, prepared from(R)-2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine, and series “b”compounds, prepared from(S)-2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine, lead to the synthesisof (L)-Fmoc-Boc-protected-neo-tryptophan “10a” and(D)-Fmoc-Boc-protected-neo-tryptophan “10b”, respectively.

FIG. 4 is a diagram depicting a scheme used to chemically synthesizeneo-tryptophan.

FIG. 5 is a graph plotting the K_(d) values obtained using the human NTreceptor for the indicated NT polypeptide analogs against the respectiveK_(d) values obtained using the rat NT receptor.

DETAILED DESCRIPTION

The invention provides a novel amino acid, neo-tryptophan, as well aspolypeptides containing this novel amino acid such as neurotensinanalogs. In addition, the invention provides neo-tryptophan derivatives,serotonin-like neo-tryptophan derivatives, and polypeptides containingsuch derivatives. The invention also provides methods for makingneo-tryptophan, neo-tryptophan derivatives, serotonin-likeneo-tryptophan derivatives, and compositions containing these compounds.Further, the invention provides methods for inducing a neurotensinresponse in a mammal as well as methods for treating a mammal having aserotonin recognition molecule.

The invention provides a novel amino acid, neo-tryptophan.Neo-tryptophan can be used to create novel polypeptides having enhancedbiological characteristics. For example, the invention providesneo-tryptophan-containing NT polypeptide analogs that exhibit enhancedbiological effects as compared to NT itself Specifically, such NTpolypeptide analogs can induce antinociception, hypothermia, thirst,weight loss, appetite suppression, and weight gain reduction. Inaddition, these NT polypeptide analogs can prevent or reduce catalepsy,such as haloperidol-induced catalepsy, and can prevent or reduce aneffect of a CNS stimulant such as apomorphine, amphetamine, and cocaine.

The incorporation of neo-tryptophan into a polypeptide sequence cancreate polypeptide analogs that exhibit increased biological activity,increased resistance to degradation by proteases (e.g.,metalloendopeptidases 24.11 and 24.16), and increased blood brainbarrier permeability. For example, neo-tryptophan can be used to makepolypeptide analogs that interact with their receptors at a higheraffinity than the natural polypeptide ligands. In addition,neo-tryptophan can be used as a novel fluorescence probe forspectroscopic studies since neo-tryptophan may have a uniquefluorescence profile.

The chemical structures for tryptophan, iso-tryptophan, tyrosine, andneo-tryptophan are provided in FIG. 1. Neo-tryptophan(2-amino-3-[1H-indolyl]propanoic acid) places the indole group oftryptophan in such a unique orientation in terms of steric andelectrostatic fields that polypeptides containing neo-tryptophan providenovel arrangements for side chain interactions. It will be appreciatedthat the term “neo-tryptophan” includes both D-neo-tryptophan andL-neo-tryptophan.

The invention provides methods for making neo-tryptophan andneo-tryptophan derivatives. Specifically, any method that results in theproduction of neo-tryptophan or a neo-tryptophan derivative is withinthe scope of the invention. For example, one method within the scope ofthe invention involves substituting the hydroxyl group of4-hydroxymethyl indole with a glycyl unit such that neo-tryptophan or aneo-tryptophan derivative is produced. In addition, FIGS. 3 and 4provide methods that can be used to synthesize both D- andL-neo-tryptophan as well as neo-tryptophan derivatives that containblocking groups. Briefly, these methods involve (a) providing2-methyl-3-nitrobenzoic acid, (b) esterifying the2-methyl-3-nitrobenzoic acid to form an esterification product, (c)reacting the esterification product with N,N-dimethylformamidedimethylacetal to produce an enamine product, (d) performing reductivecyclization on the enamine product to produce a 4-substituted indolemethyl ester, (e) protecting the indole nitrogen of the 4-substitutedindole methyl ester with a Boc group, (f) reducing the protected4-substituted indole methyl ester with DIBAL to produceN-Boc-4-hydroxymethyl indole, (g) converting the N-Boc-4-hydroxymethylindole into benzylic bromide, (h) performing SN₂ displacement of thebromide of the benzylic bromide with a carbanion to producediastereomeric bislactim products, (i) isolating one of thediastereomeric bislactim products, (j) hydrolyzing the isolateddiastereomeric bislactim product to produce an aminoester product, (k)saponifying the aminoester product to produce an N^(ind)-t-Boc aminoacid, and (l) removing the Boc group to produce neo-tryptophan. Othermethods within the scope of the invention can be easily devised by oneskilled in the art once provided with the teachings disclosed herein.

The term “neo-tryptophan derivative” as used herein refers to anycompound that has the basic structure of neo-tryptophan. Neo-tryptophanderivatives include, without limitation, neo-tryptophan having anadditional chemical group added to the glycyl group or to the indolestructure. For example, one or both of the nitrogen atoms can bemodified to contain a blocking group such as Fmoc or Boc. One suchmodification can result in a neo-tryptophan derivative having a Bocblocking group attached to the indole nitrogen atom and a Fmoc blockinggroup attached to the glycyl group nitrogen atom. Neo-tryptophanderivatives can be used during polypeptide synthesis reactions toproduce polypeptides that contain neo-tryptophan.

Any composition containing neo-tryptophan or a neo-tryptophan derivativeis within the scope of the invention. Such compositions can include,without limitation, lipids, carbohydrates, amino acids, polypeptides,nucleic acids, peptide nucleic acids, and combinations thereof.Compositions containing neo-tryptophan or a neo-tryptophan derivativecan be in an aqueous or non-aqueous form. For example, aneo-tryptophan-containing composition can contain water or saline.

Any polypeptide containing neo-tryptophan or a neo-tryptophan derivativeis within the scope of the invention. Such polypeptides can contain anysequence of natural or synthetic amino acids provided at least oneresidue is neo-tryptophan or a neo-tryptophan derivative. In otherwords, any amino acid residue within a polypeptide can be replaced withneo-tryptophan or a neo-tryptophan derivative. For example, D- orL-neo-tryptophan can be substituted for D- and L-isomers of the aromaticamino acid residues (i.e., tryptophan, tyrosine, and phenylalanine) innatural and synthetic polypeptides. Again, the incorporation ofneo-tryptophan or a neo-tryptophan derivative into an amino acidsequence can improve a polypeptide's binding affinities, selectivity,blood brain barrier permeability, and/or resistance to peptidasedegradation. Examples of polypeptides that can be modified to containneo-tryptophan or a neo-tryptophan derivative include, withoutlimitation, adrenocorticotropic hormone, angiotensin, bombesin,bradykinin, kalledin, calcitonin gene related peptide, BDNF, EGF,somatostatin, enkephalin (e.g., met-enkephalin, leu-enkephalin, andtheir derivatives), dermorphin, substance P, proctolin, isotocin,vasopressin, vasotocin, luteinizing hormone releasing hormone,neurotensin, thyrotropin releasing hormone, endomorphin-1,endomorphin-2, and morphiceptin.

The invention provides methods for making polypeptides that containneo-tryptophan and neo-tryptophan derivatives. Specifically, any methodthat results in the production of a polypeptide that containsneo-tryptophan or a neo-tryptophan derivative is within the scope of theinvention. For example, one method within the scope of the inventioninvolves linking an amino acid residue to neo-tryptophan or aneo-tryptophan derivative to form a polypeptide. For the purpose of thisinvention, the term “polypeptide” includes, without limitation,dipeptides as well as polypeptides larger than dipeptides. In addition,polypeptides containing neo-tryptophan or a neo-tryptophan derivativecan be synthesized using common polypeptide synthesis techniques withthe substitution of neo-tryptophan or a neo-tryptophan derivative whenappropriate (Morbeck D E et al., In: Methods: A Companion to Methods inEnzymology 6:191-200, Academic Press Inc., New York (1993)). Forexample, polypeptides can be synthesized using Fmoc chemistry witht-butyl-protected side chains, either individually on automatedpolypeptide synthesizers (ABI 430A or 431A) or simultaneously on amultiple polypeptide synthesizer (ACT350, Advanced Chemtech, Louisville,Ky.). Protocols concerning activation coupling times, amino aciddissolution, coupling solvents, and synthesis scale can be followedaccording to the manufacturer's instructions. Further, polypeptidescontaining neo-tryptophan or a neo-tryptophan derivative can be purifiedby, for example, reverse-phase HPLC, and then analyzed for purity by,for example, HPLC and mass spectrometry.

The term “substantially pure” as used herein refers to a molecule (e.g.,an amino acid or polypeptide) that has been separated from either thecomponents that accompany that molecule in nature or the reactionproducts (e.g., by-products) from a chemical synthesis process.Typically, a molecule is substantially pure when it is at least 50%,60%, 70%, 80%, 90%, 95%, 99%, or 99.9%, by weight, free from othercomponents or reaction products. Purity can be measured by anyappropriate method (e.g., column chromatography, mass spectrometry,polyacrylamide gel electrophoresis, or HPLC analysis).

The invention also provides serotonin-like neo-tryptophan derivatives(FIG. 2). Such serotonin-like neo-tryptophan derivatives can interactwith serotonin recognition molecules such as serotonin receptors andserotonin plasma membrane and vesicular transporters. For example,serotonin-like neo-tryptophan derivatives can be used as serotoninreceptor agonists or antagonists, inhibitors of serotonin re-uptake byplasma membrane monoamine transporters, or inhibitors of monoaminepackaging into intracellular compartments (e.g., synaptic vesicles) byvesicular monoamine transporters. In other words, the serotonin-likeneo-tryptophan derivatives provided by the invention can be used tointeract with serotonin recognition molecules in a manner such thatserotonergic conditions such as depression, anxiety, migraine,schizophrenia, eating disorders, obsessive compulsive disorders, andpanic disorders are influenced.

The chemical structure of serotonin-like neo-tryptophan derivatives isdepicted in FIG. 2. In one embodiment, R₁ can be a hydroxyl group, R₂can be an amino group, and R₃ can be either a hydroxyl group or hydrogenatom. The “X” symbol in FIG. 2 represents any chemical structure ormodification including, without limitation, blocking groups such Fmocand Boc.

Any method can be used to synthesize a serotonin-like neo-tryptophanderivative. For example, neo-tryptophan can be chemically modified usingcommon organic chemistry techniques such that a serotonin-likeneo-tryptophan derivative is produced.

In addition, the invention provides methods of inducing a neurotensinresponse in a mammal (e.g., a rodent, cow, pig, dog, cat, horse, sheep,goat, non-human primate, and human). These methods involve administeringan effective dose of a polypeptide containing neo-tryptophan or aneo-tryptophan derivative. A neurotensin response is any biologicalresponse that can be attributed to NT or a NT polypeptide analog. Forexample, a neurotensin response can be a biological response that occursafter a ligand interacts with a receptor (e.g., NTR1 and NTR2) thatbinds NT. Examples of neurotensin responses include, without limitation,antinociception, hypothermia, antipsychotic effects, loss of appetite,body weight reduction, body weight gain reduction, and increased thirst.Other examples of neurotensin responses include, without limitation, theprevention or reduction of catalepsy, such as haloperidol-inducedcatalepsy, as well as the prevention or reduction of an effect of a CNSstimulant (e.g., apomorphine, amphetamine, and cocaine). An effect of aCNS stimulant can be, for example, the climbing behavior induced byapomorphine.

The term “effective dose” as used herein refers to any amount ofcompound that induces the particular described response without inducingsignificant toxicity. For example, an effective dose of NT69L forappetite reduction can be that amount needed to cause the mammal toexhibit appetite suppression without significant toxicity. In addition,an effective dose of a particular compound administered to a mammal canbe adjusted according to the mammal's response and desired outcomes.Significant toxicity can vary for each particular patient and depends onmultiple factors including, without limitation, the patient's degree ofillness, age, and tolerance to pain.

In addition, any of the materials described herein can be administeredto any part of the mammal's body including, without limitation, brain,spinal fluid, blood stream, lungs, nasal cavity, intestines, stomach,muscle tissues, skin, peritoneal cavity, and the like. Thus, apolypeptide containing neo-tryptophan can be administered byintravenous, intraperitoneal, intramuscular, subcutaneous, extracranial,intrathecal, and intradermal injection, by oral administration, byinhalation, or by gradual perfusion over time. For example, an aerosolpreparation can be given to a mammal by inhalation. It is noted that theduration of treatment with the materials described herein can be anylength of time from as short as one day to as long as a lifetime (e.g.,many years). For example, a polypeptide containing neo-tryptophan can beadministered at some frequency over a period of ten years. It is alsonoted that the frequency of treatment can be variable. For example, apolypeptide containing neo-tryptophan can be administered once (ortwice, three times, etc.) daily, weekly, monthly, or yearly.

Preparations for administration can include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents include, without limitation, propylene glycol,polyethylene glycol, vegetable oils, and injectable organic esters.Aqueous carriers include, without limitation, water as well as alcohol,saline, and buffered solutions. Preservatives, flavorings, and otheradditives such as, for example, antimicrobials, anti-oxidants, chelatingagents, inert gases, and the like may also be present.

Any polypeptide containing neo-tryptophan or a neo-tryptophan derivativethat induces a neurotensin response can be administered to a mammal.Such polypeptides can be identified by, for example, monitoring any ofthe biological characteristics described herein before and afteradministration. In addition, a polypeptide that induces a neurotensinresponse can interact with a neurotensin receptor (e.g., a rat or humanneurotensin receptor). The term “interaction”as used herein means thattwo components specifically bind each other. Typically, any compoundthat has a binding affinity for a particular compound in thesub-millimolar range (e.g., K_(d)<1 mM) is considered to interact withthat particular compound. For example, a ligand that binds a receptorwith an affinity less than 1 mM specifically interacts with thatreceptor. Examples of polypeptides that interact with a NT receptorinclude, without limitation, NT64D, NT64L, NT65L, NT66D, NT66L, NT67L,NT69L, NT69L′, NT71, NT72, NT73, NT74, NT75, NT76, and NT77.

The invention also provides methods for treating a mammal having aserotonin recognition molecule. The methods involve administering acomposition containing neo-tryptophan, a neo-tryptophan derivative, or aserotonin-like neo-tryptophan derivative such that the compositioninteracts with a serotonin recognition molecule. The term “serotoninrecognition molecule” includes receptors as well as transporters (e.g.,plasma membrane and vesicular transporters). The interaction between thecomposition and serotonin recognition molecule can either stimulate orinhibit serotonergic activity, and thus treat conditions such asdepression, anxiety, migraine, schizophrenia, eating disorders,obsessive compulsive disorders, and panic disorders.

The invention provides a method for screening a polypeptide for in vivouse. The method involves contacting a polypeptide with a protease anddetermining whether or not the polypeptide remains intact. The term“protease” as used herein refers to any polypeptide that cleaves apeptide bond. Any source can be used to obtain proteases. For example,biological samples such as blood and intestinal tissue can be used as asource of protease. In addition, any method can be used to determinewhether or not a polypeptide remains intact. For example, polyacrylamidegel electrophoresis and HPLC analysis can be used to determine whetheror not a polypeptide remains intact.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1

Synthesis of Neo-Tryptophan and Neo-Tryptophan-Containing Polypeptides

D- and L-neo-tryptophan were synthesized according to the schemesdepicted in FIGS. 3 and 4. Polypeptides containing neo-tryptophan weresynthesized as described elsewhere using the neo-tryptophan amino acidswhen appropriate (Morbeck D E et al., In: Methods: A Companion toMethods in Enzymology 6:191-200, Academic Press Inc., New York (1993)).Briefly, polypeptides were synthesized using Fmoc chemistry witht-butyl-protected side chains, either individually on automatedpolypeptide synthesizers (ABI 430A or 431A) or simultaneously on amultiple polypeptide synthesizer (ACT350, Advanced Chemtech, Louisville,Ky.). Protocols concerning activation coupling times, amino aciddissolution, coupling solvents, and synthesis scale were followedaccording to the manufacturer's instructions. All polypeptides werepurified by reverse-phase HPLC using a C18 column (2.2×25 cm; Vydac,Hesperia, Calif.) in 0.1% TFA/water and a gradient of 10%-60%acetonitrile in 0.1% TFA/water. A combination of analytical HPLC andmass spectrometry was used to analyze polypeptide purity.

The following methods were used for the convenient, multigram synthesisof neo-tryptophan. The Fmoc-t-Boc derivative of neo-tryptophan wasreadily incorporated into bioactive synthetic peptides using standardsolid phase synthesis. The synthesis of neo-tryptophan featured the useof Schöllkopf chiral auxiliary reagents for chirality induction during akey step. For convenience, alpha-numerical designations are used todescribe specific compounds used in the synthesis steps depicted in FIG.4. In addition, a description of these compounds and the reactionprocedures are provided.

In general terms, as depicted in FIG. 4, the enantiomeric synthesis ofneo-tryptophan 1 a and neo-tryptophan derivative 1 b began with2-methyl-3-nitrobenzoic acid 2 which, after esterification, was followedby reaction with N,N-dimethylformamide dimethylacetal to furnish theenamine 4. Reductive cyclization using H₂/Pd—C gave the 4-substitutedindole methyl ester 5. Protection of the indole nitrogen of the4-substituted indole methyl ester 5 with the tert-butoxycarbonyl (Boc)group and reduction of the resulting ester with DIBAL proceededuneventfully to give N-Boc-4-hydroxymethyl indole 6. It is noted thatthe indole nitrogen was initially protected with a benzyl orcarbobenzyloxy (Cbz) group but these groups turned out to be problematiclater in the synthetic sequence. Specifically, the indole nitrogenbenzyl group could not be removed with catalytic hydrogenolysis, and theCbz group gave low yields due to its partial removal during thereduction of the methyl ester moiety with DIBAL. Next, conversion to thebenzylic bromide 7 with phosphorus tribromide in ether was followed bythe key SN₂ displacement of the bromide with the carbanion derived fromcommercially available (R)-Schöllkopf reagent,(2R)-2-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine, to provide thediastereomeric bislactim products 8 a and 8 b in 10:1 diastereomericexcess in 67% yield. The desired diastereomer 8 a was readily isolatedby flash silica gel chromatography and then subjected to mild acid (0.1M TFA/CH₃CN) treatment that only hydrolyzed the bislactim leaving theBoc group unaffected. The resulting aminoester 9 was saponified toproduce N^(ind)-t-Boc neo-tryptophan 10. It is noted that the TFA-saltof the aminoester 9 was easily isolated in pure form by extraction withmethylene chloride leaving the corresponding (D)-valine aminoester inthe aqueous phase. Finally, the amino nitrogen was protected withflourenylmethoxycarbonyl (Fmoc) to form Fmoc/Boc neo-tryptophanderivative 1 b. The Fmoc/Boc neo-tryptophan derivative 1 b wasconveniently incorporated into bioactive peptides using commonlyemployed solid phase synthesis methods. For spectral, chemical, andoptical characterization, the t-Boc group of N^(ind)-t-Bocneo-tryptophan 10 was removed to furnish neo-tryptophan 1 a. Followingthe above protocol, multigram synthesis of enantiopure Fmoc/Bocneo-tiyptophan derivative 1 b was achieved. Since the S-enantiomer ofthe Schöllkopf reagent is commercially available, the(R)-2-amino-3-(1H-indolyl)propanoic acid was similarly synthesized.

The doubly protected neo-tryptophan 1 b was readily incorporated intonovel biologically active neurotensin analogs by conventionalFmoc-related automated solid phase chemistry. In addition,neo-tryptophan was incorporated into other polypeptides havingbiological and therapeutic interest including angiotensin, bradykinin,and Leu-enkephalin (Table I).

TABLE I Amino acid sequences of NT, angiotensin, bradykinin, andleu-enkephalin polypeptides and polypeptide analogs. SequencePolypeptide 1 2 3 4 5 6 7 8 9 10 11 12 13 NT p-Glu L-Leu L-Tyr L-GluL-Asn L-Lys L-Pro L-Arg L-Arg L-Pro L-Tyr L-Ile L-Leu NT(8-13) L-ArgL-Arg L-Pro L-Tyr L-Ile L-Leu NT(9-13) L-Arg L-Pro L-Tyr L-Ile L-Leu NTWL-Arg L-Arg L-Pro L-Trp L-Ile L-Leu NT L-Arg L-Arg L-Pro L-Tyr tert-LeuL-Leu (tert-Leu) Eisai N-methyl- L-Lys L-Pro L-Trp tert-Leu L-Leu ArgNT2 D-Lys L-Arg L-Pro L-Tyr L-Ile L-Leu NT24 “27” L-Arg D-Orn^(&) L-ProL-Tyr L-Ile L-Leu NT34 L-Arg L-Arg L-Pro L-3,1′- L-Ile L-Leu Nal^(#)NT64D L-Arg L-Arg L-Pro D-neo- L-Ile L-Leu Trp NT64L L-Arg L-Arg L-ProL-neo- L-Ile L-Leu Trp NT65L L-Arg L-Arg L-Pro L-neo- tert-Leu L-Leu TrpNT66D D-Lys L-Arg L-Pro D-neo- tert-Leu L-Leu Trp NT66L D-Lys L-ArgL-Pro L-neo- tert-Leu L-Leu Trp NT67L D-Lys L-Arg L-Pro L-neo- L-IleL-Leu Trp NT69L N-methyl- L-Lys L-Pro L-neo- tert-Leu L-Leu Arg TrpNT69L′ N-methyl- L-Arg L-Pro L-neo- tert-Leu L-Leu Arg Trp NT71N-methyl- DAB^($) L-Pro L-neo- tert-Leu L-Leu Arg Trp NT72 D-Lys L-ProL-neo- tert-Leu L-Leu Trp NT73 D-Lys L-Pro L-neo- L-IIe L-Leu Trp NT74DAB L-Pro L-neo- tert-Leu L-Leu Trp NT75 DAB L-Pro L-neo- L-Ile L-LeuTrp NT76 L-Arg D-Orn L-Pro L-neo- L-Ile L-Leu Trp NT77 L-Arg D-Orn L-ProL-neo- tert-Leu L-Leu Trp Angiotensin Asp Arg Val Tyr Ile His Pro PheAngl Asp Arg Val L-neo- Ile His Pro Phe Trp Bradykinin Arg Pro Pro GlyPhe Ser Pro Phe Arg Brdyl Arg Pro Pro Gly L-neo- Ser Pro Phe Arg TrpLeu- Tyr Gly Gly Phe Leu enkephalin Lenkl L- Gly Gly Phe Leu neo- Trp*Tsuchiya Y et al., (1989) European Patent Application 89104302.8;^(#)naphthalylalanine; ^($)diaminobutyric acid; ^(&)D-ornithine

The reagents and conditions used during the steps indicated in FIG. 4can be summarized as follows: (a) K₂CO₃, MeI, DMF, RT (100%); (b)N,N-dimethylformamide dimethylacetal, DMF, 120° C.; (c) H₂, 10% Pd—C(cat), MeOH, RT, 50-55 Psi, benzene (67% over 2 steps); (d) (Boc)₂O,CH₃CN, DMAP (cat), RT (100%); (e) DIBAL-H, CH₂Cl₂/ether, −78° C. (88%);(f) PBr₃, ether/CH₂Cl₂ (95%); (g)(2R)-2-isopropyl-3,6-dimethoxy-2,5-dihydropyrazine/BuLi, THF, −78° C.,then 7 (67%); (h) 0.1 M aq. TFA, CH₃CN, RT, (100% overall); (i) LiOH,H₂O, THF/H₂O, RT (62%); (j) TFA/CH₂Cl₂, RT; (k) Fmoc-Suc, 10% NaHCO₃,acetone, 0° C.-RT, (72%).

The following section provides a detailed description of the chemicalsteps used to synthesize neo-tryptophan. In addition, the nuclearmagnetic spectra (¹H, ¹³C) described herein were measured with a BrukerWH-300 instrument (¹H frequency 300 MHZ, ¹³C frequency 75 MHZ) in thesolvent noted. ¹H chemical shifts are expressed in parts per milliondownfield from Me₄Si used as internal standard. Melting points (mp.)were taken with a GallenKamp instrument and are uncorrected. The columnchromatographic separations were performed with ‘J. T. Baker’ Silica gel(40 μm). Anhydrous DMF was obtained from Aldrich Chemicals.Tetrahydrofuran (THF) and diethyl ether were distilled over sodiumbenzophenone ketyl before use. Methylene chloride was distilled overcalcium hydride or P₂O₅. Acetonitrile was reagent grade obtained fromE.M. SCIENCE, and was used without further drying. Ethyl acetate andhexane were reagent grade, and used as received. The purity of allcompounds was shown to be >95% by TLC as well as by high field ¹H NMRand ¹³C NMR (300 and 75 MHZ Brucker instrument). Optical rotations weretaken with a 241-Perkin Elmer Polarimeter (Na lamp). IR spectra weremeasured with a 2020 GALAXY Series FT-IR (Mattson Instruments).

With reference to FIG. 4, DMF (130 mL) was added to a well mixed2-methyl-3-nitrobenzoic acid 2 (50 g, 0.28 mol) and KHCO₃ (84 g, 0.84mol) solution. Since the mixture became highly viscous, it was heated to40° C. with manual shaking. Iodomethane (79 g, 0.56 mol) was added viasyringe after the gas evolution had ceased. The resulting orange coloredsolution was stirred for 12 hours at room temperature. The reactionmixture was poured into water (800 mL), and the resulting precipitatecollected by filtration and dried over P₂O₅ to give pure methyl2-methyl-3-nitrobenzoate 3 (56 g, 100%) as a white solid: mp. 64.2-65.5°C.; ¹H-NMR (CDCl₃) δ 8.00 (d, J=7.8 Hz, 1H), 7.85 (d, J=8.0 Hz, 1H),7.39 (t, J=8.0 Hz, 1H), 3.95 (s, 3H), 2.63 (s, 3H); IR (KBr, cm⁻¹) 1724,1548, 1279; MS (EI): 195 (M⁺).

A solution of methyl 2-methyl-3-nitrobenzoate 3 (20 g, 0.1 mol) andN,N-dimethylformamide dimethyl acetal (40 mL, 0.3 mol) in DMF (50 mL)was stirred at 120° C. under nitrogen for 12 hours. The solution becamedeep red. The excess amount of N,N-dimethylformamide dimethyl acetal andDMF was distilled off under reduced pressure to give crude enamine 4that was directly used in the next step without purification.

The crude enamine 4 was dissolved in anhydrous benzene (250 mL). Pd/C(10%, 2.8 g) was added to this solution, and the resulting mixture washydrogenated at 55 psi. Warming was observed at the start of thereaction. The deep red mixture became dark gray after 12 hours at roomtemperature. Pd/C was filtered off over Celite, and the filtrate wasconcentrated under reduced pressure. Chomatography on silica gel (ethylacetate/hexanes: 30:70 v/v, Rf=0.55) afforded methyl1H-4-indolecarboxylate 5 (11.8 g, 67%) as a light yellow solid: mp.67.5-69.0° C.; ¹H-NMR (CDCl₃) δ 8.40 (s, 1H), 7.93 (d, J=8.3 Hz, 1H),7.60 (d, J=8.9 Hz, 1H), 7.36 (t, J=3.0 Hz, 1H), 7.26 (t, J=3.7 Hz, 1H),7.26-7.18 (m, 1H), 4.0 (s, 3H): IR (KBr, cm⁻¹) 3322, 1705, 1279; MS(ESI): 176 (M⁺+1).

Di-tert-butyl dicarbonate (14.7 g, 67.4 mmol) and DMAP (0.2 g) was addedto a solution of methyl 1H-4-indolecarboxylate 5 (11.8 g, 67.4 mmol) inacetonitrile (50 mL). The mixture was stirred at room temperature for 12hours. Some bubbling was observed. Solvent was removed under reducedpressure to give a residue that was redissolved in ethyl acetate (200mL). The solution was washed sequentially with cold 1N HCl (80 mL),water (50 mL), and brine (50 mL), and then dried (MgSO₄). The solventwas removed under reduced pressure to give pure 1-(tert-butyl) 4-methyl1H-1,4-indoledicarboxylate (18.5 g, 100%) as a light yellow oil: ¹H-NMR(CDCl₃) δ 8.41 (d, J=8.2 Hz 1H), 7.98 (d, J=7.7 Hz, 1H), 7.71(d, J=3.7Hz, 1H), 7.36 (t, J=7.9 Hz, 1H), 7.28 (d, J=3.8 Hz, 1H), 3.98 (s, 3H),1.68 (s, 9H); ¹³C-NMR (CDCl₃) δ 187.3, 149.4, 135.9, 130.5, 127.8,125.4, 123.5, 121.9, 119.7, 107.8, 84.1, 51.8, 28.1; IR (KBr, cm⁻¹)1703, 1603, 1283, 1146; MS (ESI): 276 (M⁺+1).

DIBAL (1.0 M in CH₂Cl₂, 163 mmol) was added at −78° C. in 30 minutesunder nitrogen to a solution of 1-(tert-butyl) 4-methyl1H-1,4-indoledicarboxylate (18.5 g, 67 mmol) in ether (150 mL). Stirringwas continued at this temperature for another 30 minutes at which pointthe reaction was quenched with saturated citric acid at −78° C. Aprecipitate immediately formed that, after warming to room temperature,was acidified to pH 1 with 1N HCl and extracted with ethyl acetate(3×200 mL). The combined extracts were washed sequentially with water(100 mL), and brine (200 mL), and then dried (MgSO₄). The solvent wasevaporated under vacuum to give a residue that was purified bychomatography on silica gel (ethyl acetate/hexanes: 30/30 v/v, Rf=0.45)yielding tert-butyl 4-(hydroxymethyl) 1H-1-indolecarboxylate 6 (14.5 g,88%) as a light yellow solid: mp. 62.9-64.1° C. ¹H-NMR (CDCl₃) δ 8.09(d, J=8.1 Hz 1H), 7.6 (d, J=3.7 Hz, 1H), 7.28 (t, J=7.5 Hz, 1H), 7.20(d, J=7.3 Hz, 1H), 6.69 (d, J=3.7 Hz, 1H), 4.90 (s, 2H), 2.01 (s, 1H),1.67 (s, 9H); ¹³C-NMR (CDCl₃) δ 149.7, 135.3, 132.7, 128.8, 125.9,124.2, 121.2, 114.8, 105.2, 83.7, 63.4, 28.1; IR (KBr, cm⁻¹) 3364, 1732,1130; MS (ESI): 248 (M⁺+1).

PBr₃ (2.4 mL. 25.8 mmol) was added dropwise at 0° C. under nitrogen to astirred solution of tert-butyl 4-(hydroxymethyl) 1H-1-indolecarboxylate6 (6.0 g, 24.3 mmol) in ether (80 mL) and CH₂Cl₂ (20 mL) under nitrogen.The reaction was completed 30 minutes after the addition. The mixturewas poured into a cold aqueous NaHCO₃ solution (100 mL) and extractedwith ethyl acetate (3×80 mL). The combined extract was washedsequentially with water (80 mL), and brine (80 mL), then dried (MgSO₄),filtered, and finally concentrated to provide tert-butyl4-(bromomethyl)-1H-1-indolecarboxylate 7 (6.3 g, 84%) as an oil whichwas immediately taken to the next step.

n-BuLi (2.5 M in hexane) was added dropwise via syringe to a solution of(2R)-2-isopropyl-3,6-dimethoxy-1,5-dihydropyrazine (3.8 g, 20.4 mmol) inTHF (70 mL) under nitrogen at −78° C. The carbanion was allowed to formfor 10 minutes at the same temperature, at which point a solution oftert-butyl 4-(bromomethyl)-1H-1-indolecarboxylate 7 in THF (40 mL) wasadded in a dropwise fashion. The reaction proceeded to completion in onehour at −78° C. Saturated aqueous NH₄Cl (80 mL) was added at −78° C.,and the THF was evaporated under reduced pressure. The aqueous phase wasextracted with ethyl acetate (3×80 mL). The combined extracts werewashed with brine (100 mL), dried (MgSO₄), and concentrated. The residuewas purified on silica gel column (ethyl acetate/hexanes: 5/95 then10/90 v/v, Rf=0.60 for the major product) to afford tert-butyl4-{[(2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydro-2-pyrazinyl]methyl}-1H-1-indolecarboxylate8 a (5.8 g, 67%) as a colorless oil: [α]_(D) ²⁵=+26.7 (c=14.8 mg/mL.CHCl₃); ¹H-NMR (CDCl₃) δ 7.98 (d, J=8.2 Hz 1H), 7.54 (s, 3H), 7.17 (t,J=7.8 Hz, 1H), 6.96 (d, J=7.3 Hz, 1H), 6.69 (d, J=4.0 Hz, 1H), 4.45-4.38(m, 1H), 3.69 (s, 3H), 3.61 (s, 3H), 3.45-3.20 (m, 3H), 2.15-2.05 (m,1H), 1.67 (s, 9H), 0.91 (d, J=6.8 Hz, 3H), 0.58 (d, J=6.8 Hz, 3H);¹³C-NMR (CDCl₃) δ 163.7, 162.2, 149.8, 134.9, 131.0, 130.0, 125.0,124.0, 121.8, 113.3, 106.5, 83.4, 60.1, 56.7, 52.3, 52.1, 36.9, 31.1,28.2, 18.9, 16.4; IR (KBr, cm⁻¹) 1734, 1696, 1346, 1128; MS (ESI): 414(M⁺+1).

TFA (0.15 N, 24 mmol) was added to a solution of tert-butyl4-{[(2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydro-2-pyrazinyl]methyl}-1H-1-indolecarboxylate8 a (3.5 g, 8.6 mmol) in acetonitrile (95 mL). The mixture was purgedwith nitrogen and stirred for 12 hours at room temperature. Theacetonitrile was evaporated, and the water phase extracted with CH₂Cl₂(5×60 mL). The combined extract was washed sequentially with water(3×100 mL), and brine (80 mL), and then dried (MgSO₄). Filtration andevaporation of the solvent left tert-butyl4-[(2S)-2-amino-3-methoxy-3-oxopropyl]-1H-1-indolecarboxylate 9 (2.7 g,98%) as a colorless oil: [α]_(D) ²⁵=+17.6 (c=12.8 mg/mL, CHCl₃); ¹H-NMR(CDCl₃) δ 8.06 (d, J=8.3 Hz 1H), 7.61 (d, J=3.7 Hz, 1H), 7.26 (t, J=7.6Hz, 1H), 7.06 (d, J=3.7 Hz, 1H), 3.84 (dd, J=5.1, 8.1 Hz, 1H), 3.70 (s,3H), 3.36 (dd, J=5.1, 13.5 Hz, 1H), 3.06 (dd, J=8.2, 13.6 Hz, 1H), 1.67(s, 9H), 1.46 (s 2H); ¹³C-NMR (CDCl₃) δ 175.4, 149.7, 135.2, 130.2,129.5, 125.8, 124.3, 114.0, 105.3, 83.7, 55.5, 52.0, 38.5, 28.1; IR(neat, cm⁻¹) 3383, 1732, 1346, 1155; MS (ESI): 319 (M⁺+1).

LiOH.H₂O (980 mg, 26 mmol) dissolved in H₂O (100 mL) at room temperaturewas added to a solution of tert-butyl4-[(2S)-2-amino-3-methoxy-3-oxopropyl]-1H-1-indolecarboxylate 9 (2.7 g,8.5 mmol) in THF (200 mL). The reaction was (close monitoring by TLC)judged complete after 10 minutes. After neutralizing with 1N HCl (30mL), the THF and most of the water were evaporated in vacuo. Theprecipitated product N^(ind)-t-Boc neo-tryptophan((2S)-2-amino-3-[1-(tert-butoxycarbonyl)-1H-4-indolyl]propanoic acid) 10(1.8 g, 62%) was collected by filtration, and dried over P₂O₅ under highvacuum: mp. 169.5-171.2° C. (dec). [α]_(D) ²⁵=−9.85 (c=6.6 mg/mL, EtOH);¹H-NMR (DMSO-d₆) δ 7.89 (d, J=8.2 Hz 1H), 7.69 (d, J=3.8 Hz, 1H), 7.27(t, J=7.7 Hz, 1H), 7.14 (d, J=7.5 Hz, 1H), 6.96 (d, J=3.8 Hz, 1H), 3.99(t, J=6.6 Hz, 1H), 3.45-3.26 (m, 2H), 1.63 (s, 9H); ¹³C-NMR (DMSO-d₆) δ170.3, 149.1, 134.6, 130.0, 128.0, 126.0, 124.3, 123.8, 113.7, 105.8,83.8, 53.3, 33.5, 27.6; IR (KBr, cm⁻¹) 3432, 3179, 1734, 1603, 1051; MS(ESI): 305 (M⁺+1).

A mixture of N^(ind)-t-Boc neo-tryptophan 10 (1.8 g, 5.28 mmol) in 10%aqueous NaHCO₃ (30 mL) was stirred for one hour at room temperature.After adding a solution of Fmoc-Suc (1.9 g, 5.55 mmol) in acetone (30mL) to this mixture, the resulting mixture was stirred for 12 hours atroom temperature. Acetone was evaporate under reduced pressure. Theaqueous phase was acidified to pH 5 with 1N HCl, and extracted withethyl acetate (3×60 mL). The combined extracts were washed with brine(80 mL), then dried (Na₂SO₄), and concentrated. The resulting residuewas purified on silica gel (MeOH/CH₂Cl₂: 5/95 v/v. Rf=0.3) as a whitesolid yielding the Fmoc/Boc derivative of neo-tryptophan((2S)-3-[1-(tert-butoxycarbonyl)-1H-4-indolyl]-2-{[(9H-fluorenylmethoxy)carbonyl]amino}propanoicacid) 1 b: mp. 92.1-93.8° C. [α]_(D) ²⁵=+5.5 (c=3.6 mg/mL, HCCl₃);¹H-NMR (DMSO-d₆) δ 7.93 (d, J=8.2 Hz 1H), 7.87 (d, J=7.6 Hz, 2H), 7.78(d, J=8.7 Hz, 1H), 7.67 (d, J=6.0 Hz, 1H), 7.59 (dd, J=7.5, 10.6 Hz,2H), 7.45-7.36 (m, 2H), 7.36-7.19 (m, 3H), 7.15 (d, J=7.2 Hz, 1H), 6.85(d, J=3.6 Hz, 1H), 4.34-4.23 (m, 1H), 4.23-4.10 (m, 2H), 3.41-3.32 (m,2H), 3.20-3.08 (m, 1H), 1.62 (s, 9H); ¹³C-NMR (DMSO-d₆) δ 173.4, 156.0,149.2, 143.9, 140.8, 134.6, 130.7, 129.9, 127.8, 127.2 126.0, 125.4,124.3, 123.6, 120.2, 113.3, 105.8, 84.0, 65.8, 55.2, 46.6, 34.2, 27.8;IR (KBr, cm⁻¹) 3308, 1703, 1346, 1128; MS (ESI): 563 (M+K⁺), 549(M+Na⁺).

A solution of N^(ind)-t-Boc neo-tryptophan 10 (10 mg, 0.03 mmol) in TFA(1 mL) and CH₂Cl₂ (2 mL) was stirred for 90 minutes. Solvent wasevaporated under reduced pressure. The residue was purified on reversephase HPLC on a Vydak C₈ column (15-20 μm particle size, 250×22 mm i.d)using a gradient of 10% B to 90% B in 30 minutes (buffer A: 0.1% TFA inH₂O; buffer B: 80% CH₃CN in buffer A; UV detection at λ_(max) 220 nm;Flow rate 8 mL/min) to give neo-tryptophan((2S)-2-amino-3-(1H-4-indolyl)propanoic acid) 1 a as a trifluoroacetatesalt: mp. 110.0-111.8° C. [α]_(D) ²⁵=+31.8 (c=1.1 mg/mL, H₂O); ¹H-NMR(DMSO-d₆) δ 11.22 (s, 1H), 8.27 (s, 3H), 7.42-7.34 (m, 2H), 7.04 (t,J=7.6 Hz 1H), 6.87 (d, J=7.1 Hz, 1H), 6.53 (s, 1H) 4.17 (s, 1H),3.41-3.26 (m, 2H); IR (KBr, cm⁻¹) 3399, 1736; MS (ESI): 205 (M⁺+1).

Example 2

Neurotensin Receptor Binding Properties of Neo-Tryptophan-ContainingPolypeptides

CHO-K1 cells were stably transfected with nucleic acid encoding eitherthe human NTR1 or the rat NTR1, and cultured in 150 mm petri plates with35 mL of Dulbecco-modified Eagle's medium containing 100 μM minimalessential medium nonessential amino acids (GIBCO) supplemented with 5%(v/v) FetalClone II bovine serum product (Hyclone Labs, Logan, Utah).CHO cells (subculture 9-19) were harvested at confluency. Briefly, themedium from each plate was removed by aspiration, and the cells washedwith 6 mL of 50 mM Tris-HCl (pH 7.4) and resuspended in 5-10 mL ofTris-HCl by scraping the cells with a rubber spatula. The resuspendedcells were placed into a centrifuge tube and collected by centrifugationat 300×g for five minutes at 4° C. in a GPR centrifuge (BeckmanInstruments, Fullerton, Calif.). The cellular pellet (in 50 mM Tris-HCl,1 mM EDTA, pH 7.4) was stored at −180° C. until radioligand binding wasperformed.

For binding assays, crude membranal preparations were prepared bycentrifugation of the cellular pellet at 35,600×g for ten minutes. Thesupernatant was decanted and discarded, and the cellular pellet wasresuspended in 2 mL. of Tris-HCl, 1 mM EDTA (pH 7.4) followed byhomogenization with a Brinkmann Polytron at setting 6 for ten seconds.Centrifugation was repeated as above and the supernatant was decantedand discarded. The resulting final cellular pellet was resuspended in 50mM Tris-HCl, 1 mM EDTA, 0.1% bovine serum albumin, and 0.2 mMbacitracin. Polypeptide concentration of the membranal preparation wasestimated by the method of Lowry et al. (J. Biol. Chem. 193:265-275(1951)) using bovine serum albumin as a standard.

A Biomek 1000 robotic workstation was used for all pipetting steps inthe radioligand assays as previously described (Cusack and Richelson, J.Recept. Res. 13:123-34 (1993)). Competition binding assays with [³H]NT(1 nM), varying concentrations of unlabeled NT, and polypeptide analogswere carried out with membranal preparations from the appropriate celllines. Nonspecific binding was determined with 1 μM unlabeled NT inassay tubes with a total volume of 1 mL. Incubation was at 20° C. for 30minutes. Each reaction was terminated by addition of cold 0.9% NaCl(5×1.5 mL) followed by rapid filtration through a GF/B filter strip thathad been pretreated with 0.2% polyethylenimine. Details of bindingassays are described elsewhere (Cusack et al., Mol. Pharmacol.44:1036-1040 (1993)). The data were analyzed using the LIGAND program(Munson and Rodbard, Anal. Biochem. 107:220-239 (1980)). The valuespresented for K_(d) are expressed as the geometric means±SEM (Fleming etal., J. Pharmacol. Exp. Ther. 182:339-345 (1972) and DeLean et al., Mol.Pharmacol. 21:5-16 (1982)).

Radioligand binding assays were performed using various NT analogs. Ineach case, the equilibrium dissociation constant (K_(d)) was derived forboth human NTR1 l and rat NTR1 (Table II). All polypeptides tested had aHill Coefficient close to unity, indicating that binding was to a singleclass of receptors. Substituting L-neo-tryptophan for Tyr¹¹ in NT (8-13)resulted in the most potent compound (NT64L) tested at the humanreceptor, and nearly the most potent tested at the rat receptor. Infact, the binding affinity of NT64L was in the range of that found for[L-3, 1′-Nal¹¹]NT(8-13) (NT34) at the rat receptor. NT72, apentapeptide, was found to be the least potent at both receptors. Whilesubstituting D-Lys for L-Arg⁸ in NT64L resulted in a polypeptide (NT67L)exhibiting greater resistance to peptidase degradation than NT64L, theNT67L polypeptide exhibited a binding affinity (K_(d)=0.61 nM) about sixfold lower at the human receptor than that exhibited by NT64L(K_(d)=0.09 nM). In addition, steric factors appear to influence NTreceptor binding since the results revealed a more than 30 foldreduction in binding affinity for NT64D, which contains the D-isomer ofneo-tryptophan, and for NTW, which contains the natural isomer oftryptophan, when compared to NT64L.

TABLE II Comparison of binding affinity for NT analogs at human and ratNT receptors. K_(d) [nM] Polypeptide hNTR rNTR NT64L 0.09 ± 0.01 (3)0.10 ± 0.01 (5) NT(8-13) 0.14 ± 0.01 (4) 0.16 ± 0.01 (3) NT65L 0.32 ±0.01 (5) 0.075 ± 0.004 (3) NT67L 0.61 ± 0.06 (3) 0.21 ± .02 (10) NT2 1.0± 0.1 (6) 0.8 ± 0.1 (3) NT69L 1.55 ± 0.09 (5) 0.82 ± 0.07 (4) NT71 1.8 ±0.1 (4) 0.22 ± 0.03 (8) NT(1-13)  1.97 ± 0.07 (130)  2.39 ± 0.08 (99)NTW 3.2 ± 0.3 (3) 0.34 ± 0.03 (3) NT64D 3.3 ± 0.4 (3) 3.8 ± 0.4 (3)NT66L 3.7 ± 0.4 (3)  0.85 ± 0.09 (14) NT34 5.8 ± 0.6 (4) 0.046 ± 0.003(3) NT(tert-Leu) 13.2 ± 0.5 (4)  19.8 ± 0.4 (4)  NT(9-13) 30 ± 2 (3)  46± 4 (3)  NT75 34 ± 1 (5)  10.0 ± 0.4 (5)  NT73 45 ± 3 (3)  32 ± 6 (6) Eisai 95 ± 9 (12) 5.4 ± 0.6 (8) NT66D 210 ± 20 (10) 77 ± 9 (8)  NT74 360± 10 (3)  160 ± 40 (3)  NT72 640 (2) 270 ± 30 (5)  Values are geometricmean ± SEM, n value is in parenthesis; K_(d) = equilibrium dissociationconstant in CHO-K1 membranes; n.d. = no data.

In general, substitution of L-Ile¹² with tert-Leu, a substitute for thenatural amino acid leucine, lowered the binding affinity of the NTanalogs when compared to their counterparts containing L-Ile¹². Forexample, NT(tert-Leu) was about 100 fold less potent at both the human(K_(d)=13.2 nM) and rat (K_(d)=19.8 nM) receptors than was NT(8-13)(K_(d)=0.14 nM and 0.16 nM at human and rat receptors, respectively). Asmaller decrease (3 fold) in binding affinity resulted when L-Ile¹² wasreplaced with tert-Leu as observed between NT64L and NT65L.

The Eisai compound was found to be almost 20 fold weaker at the human NTreceptor when compared to its binding affinity at the rat receptor.Substituting L-Trp¹¹ in Eisai with L-neo-tryptophan to give NT69Lresulted in a 60 fold increase in binding affinity at the humanreceptor, but only a 6 fold increase at the rat receptor.

Other modifications included substitutions in the sequence of NT(9-13).Briefly, NT(9-13) was found to have low affinity for NT receptors. Infact, NT(9-13) was over 200 fold weaker at the human and rat receptorsthan was NT(8-13). Of the pentapeptides tested (NT72, NT73, NT74, andNT75), NT75 was found to be the most potent (K_(d)=34 nM and 10 nM atthe hNTR1 and rNTR1, respectively). These pentapeptides, however, werenot more potent than NT(9-13). Again, substitution of Ile¹² withtert-Leu caused a several fold reduction in binding affinity as observedwhen NT75 is compared to NT74, and when NT73 is compared to NT72.

The K_(d) values obtained using the human NT receptor for the various NTanalogs were plotted against the respective K_(d) values obtained usingthe rat receptor (FIG. 5). This analysis revealed a strong correlation(R=0.88, P<0.0001) between the binding affinity at human and rat NTreceptors. In addition, the line of identity (dotted line havingslope=1) revealed that most NT analogs have a higher binding affinityfor the rat receptor than for the human receptor, while some have asimilar binding affinity for both receptors. No NT analog exhibited ahigher binding affinity for human NTR1 than for rat NTR1.

In summary, every tested NT analog containing L-neo-tryptophan exhibitedan increase in binding affinity over similar NT analogs (e.g., NTW vs.NT64L, NT2 vs. NT67L, and Eisai vs. NT69L). Thus, the addition ofL-neo-tryptophan contributed significantly to increasing the potency ofNT analogs.

Example 3

PI Turnover Properties of Neo-Tryptophan-Containing Polypeptides

To measure PI turnover, intact CHO-K1 cells were harvested at about 80%confluency. Cells were detached from the petri plates by removal ofculture medium followed by incubation of the cellular monolayer for 20minutes at 37° C. with gentle shaking in a modified Puck's D₁ solutioncontaining 2 mM EGTA. The details of assaying the relative changes in PIturnover in intact cells using a radioactively labeled precursor wasdescribed elsewhere (Pfenning and Richelson, In: Methods inNeurotransmitter Receptor Analysis Eds: Yamamura H I, S J Enna and M JKuhar, pp.147-175, Raven Press, New York (1990)). Briefly, intact CHOcells were prelabeled with D-myo-[³H]inositol (18.3 Ci/mmol) in thepresence of lithium chloride (final concentration, 10 mM). Cells werethen stimulated with NT or the appropriate NT analog, and the amount of[³H]inositol 1-phosphate ([³H]IP₁) produced by the cells isolatedchromatographically on Dowex 1-X8 (200-400 mesh). For the experimentsdescribed herein, the stimulation time was 30 minutes, and the number ofCHO cells per assay tube was 1.5×10⁵. The presented EC₅₀ values areexpressed as the geometric means±SEM (Fleming et al., J. Pharmacol. Exp.Ther. 182:339-345 (1972) and DeLean et al., Mol. Pharmacol. 21:5-16(1982)).

Each tested NT analog was functionally coupled to PI turnover asdetermined using intact CHO-K1 cells (Table III). The most potent NTanalog tested at the human NTR1 was NT67L (EC₅₀=0.83 nM). SubstitutingIle¹² in NT67L with tert-Leu to give NT66L did not improve potency atthe human NTR1 (EC₅₀=10 nM). For NT69L, an EC₅₀ of 2.3 nM and 1.3 nM atthe hNTR1 and rNTR1, respectively, was observed.

TABLE III Comparison of PI turnover results for NT analogs at human andrat NT receptors. PI Turnover EC₅₀ [nM] Polypeptide hNTR rNTR NT64L  2.8± 0.5 (4) 2.3 ± 0.2 (3) NT(8-13)  1.5 ± 0.1 (3) n.d. NT67L  0.83 ± 0.09(3) 13 ± 2 (3)  NT69L  2.3 ± 0.5 (3) 1.34 ± 0.02 (3) NT(1-13)  5.0 ± 0.3(39)  6.8 ± 0.4 (10) NTW 110 ± 20 (5) n.d. NT66L 10 ± 2 (4) 1.9 ± 0.4(3) NT34 130 ± 20 (3) 2.8 ± 0.2 (4) NT(9-13) 380 ± 50 (3) n.d. Eisai 300± 20 (4) 3.1 ± 0.4 (3) Values are geometric mean ± SEM, n value is inparenthesis; EC₅₀ = concentration of compound needed to stimulate 50% ofmaximum PI response in intact CHO-K1 cells; n.d. = no data.

In general, the EC₅₀ values observed for the various tested analogs weresimilar at rat NTR1 (in the 1-13 nM range). At the human NT receptor,however, the EC₅₀ values were quite different (in the 1-300 nM range forthe hexapeptides tested). These results may reflect the size of thebinding site at the human NTR1 and the conformation of the NTanalog-receptor complex, with the human receptor being much lesstolerant of size and steric changes of the ligand. In addition, theEisai analog was nearly 100 fold weaker at the human NT receptor than atthe rat NT receptor (EC₅₀ of 300 nM at human NTR1, and 3.1 nM at ratNTR1). This difference was less striking than that found for therespective affinities in radioligand binding experiments with membranalpreparations from these cells (Table II). In addition, comparing resultsobtained using the Eisai analog to those obtained using NT69L revealedan improvement for NT69L in the potency at PI turnover of about 130 foldat human NTR1 and about two fold at rat NTR1. Thus, these resultsindicate that the addition of L-neo-Trp to NT analogs significantlyinfluences their pharmacology and biochemistry at human NTR1.

Example 4

Degradation Properties of Neo-Tryptophan-Containing Polypeptides

The following experiments determined the stability of the novelpolypeptides containing neo-tryptophan in rat and human plasma as wellas rat intestinal preparations. Whole blood was collected into tubescontaining heparin (200 units/mL), and placed on ice. Samples were spunat 500×g for ten minutes. The supernatant was recovered and frozen at−20° C. overnight. The samples were thawed at room temperature and spunat 500×g for ten minutes. After recovery, the supernatant was filteredthrough 0.2 μm syringe filter.

For degradation studies, ultrapure water that had been filtered twicethrough 0.02 μm filters was used. Each polypeptide to be tested wasresuspended in this water at a concentration of 1 mg/ml. The filteredplasma was diluted 1:1 with the filtered H₂O (50% v/v). Then, 500 μL ofdiluted plasma was combined with 500 μL of the polypeptide solution (1mg/mL), and vortexed. The final concentration of polypeptide was 0.5mg/mL in 25% plasma (v/v).

An initial time point was taken at 0° C. before placing theplasma/polypeptide sample in a 37° C. water bath. At each time point, 50μL of the plasma/polypeptide sample was removed and combined with 1.050mL of filtered H₂O, representing a 1:22 dilution. Thus, the final amountof material injected into the HPLC represents 22.7 μg of polypeptide in1.14% plasma. Peak area was recorded at each time point and compared tothe plasma/polypeptide sample at zero time at 0° C. Values are expressedas the percent of peak area at zero time.

The HPLC conditions were as follows: C-18 column; flow rate equal to 3mL/minute; and the gradient equal to 10-90% B in 50 minutes, whereA=TFA, 0.1% and B=TFA 0.1% in 80% acetonitrile.

For intestinal preparations, rats were sacrificed by decapitation, andthe small intestines removed. The tissue was washed with ice-cold PBS(10 mM, pH 7.4). About 2 mg of intestinal tissue was added to about 10mL of PBS and homogenized using a Brinkman polytron homogenizer (setting6). After homogenization, 10 mL of PBS was added to 10 mL of theintestinal homogenate. The mixture was then centrifuged at 500×g for 15minutes at 4° C. The supernatant was removed and stored at −20° C. untiltesting. Immediately before incubation with the polypeptide, theintestinal preparation was filtered through a 0.2 μm filter. Testingprocedures were similar to those for the plasma tests.

For the degradation studies, data were plotted in linear form as asemi-log plot with SigmaPlot for Windows Version 4.00. Linear regressionon these plots provided the parameters used to calculate the half-time(t_(1/2)) for degradation of each polypeptide. The correlationcoefficient (R) for the linear regression was taken as a measure of thegoodness-of-fit.

L-neo-tryptophan provided increased resistance to peptidases. Thehalf-life of NT2 in human plasma was determined to be about 0.85 hours(Table IV). Substituting L-Tyr¹¹ of NT2 with L-neo-tryptophan resultedin a polypeptide (NT67L) having a half-life in human plasma of about 96hours. In addition, substituting L-Trp¹¹ of Eisai with L-neo-tryptophanresulted in a polypeptide (NT69L) having a longer half-life in humanplasma. These results demonstrate that the substitution of animo acidresidues with neo-tryptophan can produce polypeptides having increasesresistance to degradation.

TABLE IV Half-life-values for Neurotensin (NT) and NT analogs. PlasmaRat Intestinal Human Rat Preparation half-time half-time half-timePolypeptide (hours) R (hours) R (hours) R NT 1.9 0.99 5.4 1.0  0.04 1.00NT(8-13) n.d. 0.22 0.98 n.d. NT2 0.85 0.71 n.d. n.d. NT64L 1.4 0.96 n.d.n.d. NT65L 1.1 1.00 n.d. n.d. NT66L 170 0.94 350 0.94 n.d. NT67L 96 0.97130 0.95 0.77 0.94 NT69L 500 0.90 250 0.88 4.1 0.91 NT71 n.d. 260 0.725.1 0.98 NT72 3000 0.19 n.d. 54 0.99 NT73 300 0.58 n.d. 0.83 0.99 NT74n.d. 110 1.00 n.d. NT75 n.d. 1.6 0.93 0.27 0.96 Eisai 460 0.91 n.d. 220.96

These degradation studies also revealed some difference between humanand rat plasma For example, NT had a half-life in human plasma of aboutone-third that observed in rat plasma. In general, however, thehalf-life times observed for the tested analogs correlated reasonablywell between the human and rat plasma samples (slope=0.76, R=0.97, df=3,P=0.03). In human plasma, NT, NT2, NT64L, and NT65L were the mostrapidly degraded. The remaining analogs tested all had significantlylonger half-life times in human plasma In addition, all hexapeptide NTanalogs containing a substitution for Arg⁸ and all pentapeptide NTanalogs containing a substitution for Arg⁹ were found to besubstantially resistant to degradation. Similar results were obtainedusing the rat plasma

NT72 was found to be the most resistant to degradation by rat intestinalproteases, while NT67L, an analog relatively stable in plasma, wasdegraded very rapidly by rat intestinal proteases. Interestingly, NT69Lwas less stable in the rat intestinal preparation than was the Eisaianalog. Again, NT69L was found to be more resistant to human plasmaproteases than the Eisai analog. Substituting Ile¹² of NT73 withtert-Leu resulted in a polypeptide analog (NT72) exhibiting asubstantial increase of stability in the intestinal preparation whencompared to NT73.

In general, these results demonstrate that NT analogs having unnaturalamino acid substitutions at positions 8 and 12 for the hexapeptides andpositions 9 and 12 for the pentapeptides are more resistant todegradation in plasma and in the rat intestinal preparation than NTanalogs not having such substitutions.

Example 5

Antinociceptive and Hypothermic Properties of Neo-Tryptophan-ContainingPolypeptides

Male Sprague Dawley rats (Harlan, Prattville, Ala.; 150-250 g) werehouse in a temperature controlled room with a 12 hour light/dark cycle,and given water and standard rat chow. Testing occurred during the lightcycle. Hot plate measurements were performed to assess antinociception,while body temperature measurements were taken to assess hypothermia.

The baseline hot plate measurements and body temperatures weredetermined immediately prior to each experiment. Briefly, the hot platewas performed to determine pain sensitivity. Thirty minutes afteradministration of the test compound, the rat was placed on the hot plateand latency was measured. Hot plate measurements were taken on a metalsurface (15×20 cm) maintained at a temperature of 52.0±0.15° C.(Al-Rodhan et al., Brain Res. 557: 227-235 (1991), and Jensen and Yaksh,Brain Research 372:301-312 (1986)). The latency between the time the ratwas placed on the surface and the time it licked either of its hind pawswas measured. Failure to respond in 30 seconds resulted in ending of thetrial and assignment of that latency. Animals were removed immediatelyafter responding or at the cutoff latency. Hot plate tests were scoredas the percent of maximum possible effect (% MPE) and calculated usingthe following equation: % MPE=[(post-drug latency−pre-druglatency)/(cut-off−pre-drug latency)]×100; where the cut-off was 30seconds. Immediately after completion of the hot plate test, bodytemperature measurements were taken using a digital thermistor probeinserted into the rectum about 2-4 cm.

For these behavioral and physiological measurements, data were testedfor significance with a student's t-test and p<0.05 was consideredsignificant. Preference for the t-test instead of the ANOVA was givendue to the reasons cited elsewhere (O'Brien P C, Biometrics 39:787(1983)).

For intraperitoneal (ip) delivery, the test compound was injected intothe intraperitoneal cavity while control rats received an equal volumeof saline (0.9% NaCl). For nasal delivery, the test compound wasdissolved in 4 μL of sterile saline, and the rats lightly anaesthetizedwith CO₂. The rats then were held in a vertical position while 2 μL ofthe test compound were delivered to each nostril using a polyethylenegel loading tip attached to a Gilson P20 pipettor. The rats remained ina vertical position until it was clear that all the liquid had beeninhaled into the nostril. After inhalation, the rats were allowed torecover from the anesthesia which usually occurred within one minute.For subcutaneous (sc) delivery, the test compound was injected into thefold of skin at the back of the neck. Injection volumes were about 100μL. For oral deliver, a gavage device attached to a syringe thatextended into their stomachs was used to ensure complete delivery of thetest compound. The volume delivered was about 0.3 mL.

The following methods were used to deliver the test compounds directlyinto brain. Under sterile conditions, the rats were stereotaxicallyimplanted with stainless steel guide cannulae (26 gauge) into theperiaqueductal gray (PAG) region of the rat brainstem under sodiumpentobarbital anesthesia (50 mg/kg, ip) as described in detail elsewhere(Jensen and Yaksh, Brain Res. 372:301-312 (1986), and Al-Rodhan, BrainRes. 557:227-235 (1991)). The coordinates used for PAG cannulations are−5.6 mm posterior from bregma, 1.0 mm lateral from bregma, and 5.5 mmdown from the dura. The guide cannula was pre-measured to be 5.5 mm(Plastics One, Roanoke, Va.) and the internal cannula was ordered to fitbelow the pedestal with a 2.0 mm projection. The guide cannula was thenfixed to the skull using a stainless steel screw (⅛ inch) andcranioplastic cement. A stainless steel stilette was then placed in eachguide to keep it patent and free of debris. Immediately after surgery,the animals were allowed to recover before returning them to anindividual housing cage. All injections began 5-7 days after surgery. Ifany problem, such as an infection, was observed with an animal aftercannulation, then the animal was euthanized immediately by decapitation.

Intraperitoneal administration of NT64L (1 mg/kg) did not induceantinociception as measured by the hot plate test or hypothermia Wheninjected into the PAG, however, NT64L (18 nmol) induced bothantinociception and hypothermia Specifically, rats receiving NT64Lexhibited a peak % MPE value of 76% at 30 minutes, and a peak bodytemperature reduction of 2.1° C. at 30 minutes. NT and NT24“27” also didnot induce antinociception or hypothermia upon intraperitonealadministration. After PAG administration, rats receiving NT (18 nmol)exhibited a peak % MPE value of 80% at 30 minutes, and a peak bodytemperature reduction of 1.8° C. at 30 minutes, while rats receivingNT24“27” (18 nmol) exhibited a peak % MPE value of 20%, and a peak bodytemperature reduction of 1.1° C. at 30 minutes.

Intraperitoneal administration of NT66D, NT66L, NT67L, NT69L, NT71,NT72, NT73, and Eisai did induce antinociception and hypothermia (TableV).

TABLE V Antinociception and hypothermia after intraperitonealadministration of NT analogs. 1 mg/kg 5 mg/kg Peak BT Peak % Peak BTPeak % ED₅₀ ED₅₀ Polypeptide change MPE change MPE BT % MPE Eisai −2.9 @80% @ n.d. n.d. 0.26 mg/kg 0.42 mg/kg 60 min 90 min @ 30 min @ 30 min0.12 mg/kg 0.08 mg/kg @ 90 min @ 90 min NT66D* −1.0 @ 58% @ n.d. n.d.n.d. n.d. 30 min 30 min NT66L −3.0 @ 100% @ −5.0 @ 100% @ 0.45 mg/kg0.04 mg/kg 240 min 120 min 120 min 330 min @ 30 min @ 30 min 0.18 mg/kg0.02 mg/kg @ 60 min @ 60 min NT67L −1.8 @ 70% @ n.d. n.d. n.d. n.d. 120min 90 min NT69L −5.3 @ 100% @ n.d. n.d. 0.4 mg/kg 0.3 mg/kg 300 min 300min @ 90 min @ 90 min NT71 −2.0 @ 70% @ −2.4 @ 79% @ n.d. n.d. 40 min 60min 90 min 180 min NT72 n.d. n.d. −2.8 @ 100% @ n.d. n.d. 180 min 120min NT73 n.d. n.d. −0.9 @ 40% @ n.d. n.d. 90 min 120 min *NT66D wasadministered at a dose of 0.5 mg/kg instead of 1 mg/kg.

NT69L had potent and long lasting behavioral effects. Specifically,NT69L given intraperitoneally to rats at a dose of 1 mg/kg induced asignificant reduction in body temperature, reaching a peak of −5.3° C.This peak reduction of body temperature was reached about 90 minutesafter administration and remained significant up to 300 minutes aftertreatment. In addition, NT69L produced significant and long lastingantinociception. Specifically, a peak % MPE value of 100% was observedfor up to about 200 minutes after treatment. Analysis of the time coursefor NT69L-induced antinociception and hypothermia revealed that the peakantinociception effect (100% MPE) remained for almost three hours afteradministration while the hypothermia effect began to recover from thepeak value of −5° C. at about 90 minutes. One possible explanation forthese time course differences is the existence of different NT receptorsubtypes that subserve these two different behavioral responses.

The results from a dose response analysis using NT69L indicated that theED₅₀ value for body temperature lowering (hypothermia) was 0.4 mg/kg at90 minutes, while the ED₅₀ value for % MPE (antinociception) was 0.3mg/kg at 90 minutes. For NT66L, the ED₅₀ value was found to be 0.45mg/kg at 30 minutes for hypothermia, and 0.04 mg/kg at 30 minutes forantinociception. For Eisai, the ED₅₀ value was found to be 0.26 mg/kg at30 minutes for hypothermia, and 0.42 mg/kg at 30 minutes forantinociception. Although the ED₅₀ values for Eisai and theneo-tryptophan-containing NT analogs (NT66L and NT69L) may appearsimilar in degree, their effects were found to be very different. Forhypothermia, animals treated with the Eisai compound exhibited a peakreduction in body temperature of about 3° C. at about one hour. NT69Linduced a 5.3° C. reduction in body temperature that was maintained forup to five hours. For antinociception, animals treated with the Eisaicompound reached peak of about 80% MPE at 90 minutes. This peak level,however, started to drop shortly thereafter, returning to baselinelevels at about six hours after administration. The antinociceptioninduced by NT69L, however, was maintained at a significant level for upto five hours.

The effectiveness of NT analogs administered by several routes also wasanalyzed. For NT69L, the antinociception and hypothermia resultsobserved after subcutaneous administration were similar to thoseobtained after intraperitoneal administration with the exception thatthe observed effects after subcutaneous administration appeared to lagbehind the effects observed after intraperitoneal administration. Nasaladministration of NT69L also produced antinociception and hypothermia.Specifically, NT69L given nasally to rats at a dose of 5 mg/kg induced areduction in body temperature, reaching a peak of −1.4° C. at 30minutes, and induced antinociception with a peak %MPE value of 70% at 60minutes. Thus, the nasal administration of NT69L appeared to induceantinociception more effectively than hypothermia. In addition, oraladministration of NT69L (20 mg/kg) induced a reduction in bodytemperature, reaching a peak of −0.63° C. at 60 minutes, and inducedantinociception with a peak %MPE value of 11% at 30 minutes. While thehypothermia response was significant after oral administration of NT69L,the antinociceptive effect was not. Oral administration of NT66L,however, induced significant antinociception and hypothermia.Specifically, oral administration of NT66L (20 mg/kg) induced areduction in body temperature, reaching a peak of −1.4° C. at 30minutes, and induced antinociception with a peak %MPE value of 40% at 60minutes.

Example 6

Interactions Between Brain Receptors and Neo-Tryptophan-ContainingPolypeptide

In a radiolabeled competitive binding assay, NT64L, NT66L, and NT67Lwere found to compete with labeled ketanserin for binding at the5HT_(2A) receptor in human brain tissue (Table VI). Specifically, NT64Lhad a K_(d) of 6.6 μM at this receptor in a competition binding assayusing [³H]ketanserin as the radioligand. In addition, NT2, NT(8-13), andNT(9-13) were found to compete with labeled ketanserin for binding atthe 5HT_(2A) receptor in human brain tissue. L-neo-tryptophan itself,however, did not compete with labeled ketanserin for binding at the5HT_(2A) receptor. These results indicate that NT analogs can interactwith serotonin recognition molecules.

TABLE VI Binding-affinities for serotonin receptors. Kd [nM] Human BrainTissue Compound 5HT_(2A) NT69L 34600 NT66L 8700 NT67L 7400 NT(9-13) 6340NT2 6800 L-neo-Trp >100000 NT64L 6600 NT(8-13) 4400 Serotonin 680Haloperidol 61 Clozapine 9.1

Additional radiolabeled competitive binding assays were designed toassess the interaction of NT analogs and other ligands with othervarious types of receptors such as adrenergic and dopamine receptors(Table VII). These studies revealed that NT67L can interact withadrenergic α1 receptors having a K_(d) value of 6.9 μM. In addition,these studies revealed that L-neo-tryptophan itself does not bind tohuman NT receptors from CHO cells.

TABLE VII Comparison of binding at different receptors. K_(d) (nM) Humanbrain tissue adrenergic CHO cells Compound α1 α2 Muscarinic DopaminerNTR hNTR NT66L 38000 n.d. n.d. 56000 0.85 3.7  NT67L 6900 >100000 >100000 >100000    0.21 0.61 NT69L n.d.    58600 n.d.13300 0.82 1.55 L-neo-Trp n.d. n.d. n.d. n.d. n.d. >100000 Serotoninn.d. n.d. n.d. n.d. >100000 >100000 Haloperidol   17 600  >100002.6 >100000 >100000 Clozapine   19  16 8.5 211 >100000 >100000

Example 7

Neo-Tryptophan-Containing Polypeptides and CNS Stimulants

Apomorphine was used to assess the ability of NT analogs to act asneuroleptics. Briefly, male Sprague-Dawley rats were pretreatedintraperitoneally with either NT69L or saline only. Thirty minutesfollowing pretreatment, the rats received a subcutaneous injection ofapomorphine (600 μg/kg). Occasionally, the effectiveness of NT69L wasassessed by measuring antinociception and hypothermia just prior toapomorphine administration. Control animals included rats receivingNT69L followed by saline only, and rats receiving no treatment. Theapomorphine was dissolved in oxygen-free boiled 0.9% NaCl solutioncontaining 0.1% ascorbic acid and 0.1% metabisulfite to preventoxidation. The volume of injection was 1 mL/kg under the loose skin atthe back of the animal's neck. Immediately following injection, the ratswere placed in cages for observation. About 5-10 minutes later,behavioral monitoring was initiated and lasted for one hour. Climbingepisodes were measured by observing the number of times the rat moved upand down in a vertical position during a two minute observation period.Statistical analysis was done using the Student's t-test with P<0.05being considered significant. Graphs and ED₅₀ data were generated usingGraphPad Prism® software (version 2.01, GraphPad Software, Inc., SanDiego, Calif.).

Rats receiving saline followed by apomorphine exhibited distinctiveclimbing, sniffing, and licking behaviors that lasted for about 60minutes after treatment. Pretreatment with NT69L (1 mg/kg) 30 minutesbefore the apomorphine injection caused a long-lasting blockade of theclimbing behavior. This NT69L pretreatment, however, did not influencethe sniffing and licking behaviors induced by apomorphine. Controlanimals receiving no treatment or NT69L without apomorphine did noexhibit any of these behaviors. The ED₅₀ as determined by non-linearregression analysis for NT69L at 75 minutes after injection of NT69L (45minutes after injection of apomorphine) was 16 μg/kg (95% confidenceinterval; 6.1 to 44 μg/kg; R=0.94). At no dose did NT69L affect theoro-facial stereotypies. Thus, NT69L was found to be an extremely potentcompound capable of preventing the climbing behavior induced byapomorphine. These results indicate that NT69L may have clinical effectssimilar to those of an atypical neuroleptic. The site of the blockade ofapomorphine-induced climbing by NT69L does not appear to be dopamine,serotonin, or adrenergic receptors since NT69L was found to have a weakaffinity for dopamine D₂ receptors as well as 5HT_(2A) and α₂-adrenergicreceptors (Table VII).

At 5 mg/kg (ip), a dose of NT69L that completely blocked the effects ofthe apomorphine-induced climbing behavior, NT69L caused a largereduction in body temperature. Specifically, a change in bodytemperature of about −3° C. was observed 30 minutes after injection,while a change of about 4° C. was observed at 77 minutes afterinjection. At 90 minutes, the ED₅₀ for NT69L-induced body temperaturelowering was 390 μg/kg (95% confidence interval; 110 to 1400 μg/kg,R=0.98). Injection of apomorphine alone caused a more modest reductionof body temperature than did NT69L. The hypothermic effects observed inanimals receiving NT69L alone were not different from those observed inanimals receiving the combination of NT69L and apomorphine. Thus, theeffects of apomorphine and NT69L on body temperature were not additive.The fact that there was no additive effects of these compounds onhypothermia indicates a common site of action or a “ceiling” effect onbody temperature lowering. In other words, NT69L may cause the maximumbody temperature lowering possible in the animals.

The following experiments were performed to assess the ability of NT69Lto cause and influence catalepsy. Male Sprague-Dawley rats (150-250 g)or CD-1 mice (24-26 g) were housed in a temperature-controlled room ingroups of five with free access to food and water. The animals were kepton a 12 hour light/dark cycle, and all tests were performed during thelight cycle. The test for catalepsy is well established and quite simple(Munkvad et al., Brain Behav. Evol. 1:89-100(1968)). Animals receivedone of three compounds intraperitoneally: NT69L, a typical neuroleptic(haloperidol), or an atypical neuroleptic (clozapine). Thirty minutesafter injection, the animals were tested for catalepsy. For rats, thetest involved simply placing the animal's fore paws on a suspended metalbar 10 mm in diameter, 11 cm above the counter. The time elapsed untilthe animal's fore paws touch the counter was recorded. If the rat didnot drop to the counter top by four minutes, the rat was removed fromthe bar. For mice, catalepsy was scored based on the time that the micemaintained a fixed rearing posture against the side of the cage (Adamset al., Proc. Natl. Acad. Sci. USA 94:12157-12161 (1997)). The cut-offtime was 20 seconds, and each animal was tested three times for eachtime point.

NT69L at a dose that induced significant hypothermia (5 mg/kg; ip) hadno effect on catalepsy scores in rats. At the same dose, NT69L caused nocatalepsy in mice. As expected, however, haloperidol caused profoundcatalepsy at a dose (1 mg/kg) that had minimal effect on bodytemperature. In addition, the haloperidol administration did notinfluence the body temperature lowering effect of NT69L when both drugswere given to the same animal. The haloperidol-induced catalepsy wasobserved at 30 minutes and lasted for at least six hours. The ED₅₀ valuefor haloperidol-induced catalepsy was found to be 130 μg/kg (95%confidence interval: 280 to 520 μg/kg, R=0.998) at 30 minutes; 260 μg/kg(95% confidence interval: 130 to 530 μg/kg, R=1.00) at 60 minutes, and310 μg/kg (95% confidence interval: 42 to 2300 μg/kg, R=1.00) at 180minutes.

When animals were injected with NT69L (5 mg/kg; ip) 30 minutes beforeinjecting haloperidol (1 mg/kg; ip), the rats did not exhibitsignificant cataleptic behavior. When animals were injected with NT69L(5 mg/kg; ip) 30 minutes after injecting haloperidol (1 mg/kg; ip), themoderate catalepsy observed 30 minutes after the haloperidol treatmentwas reversed by 30 minutes after NT69L treatment. From the 30 minutetime point after NT69L treatment, the rats no longer exhibitedcatalepsy, and remained non-cataleptic for up to four hours from thetime of haloperidol injection. These results indicate that NT69L givenbefore haloperidol blocks haloperidol-induced catalepsy, while NT69Lgiven after haloperidol reverses the catalepsy induced by haloperidol.

To determine the ED₅₀ value of NT69L for the reversal of the catalepticeffects induced by haloperidol, animals were given haloperidol (120μg/kg; ip) followed by varying doses of NT69L (ip) 40 minutes later.Catalepsy was scored at 130 minutes. The ED₅₀ value for NT69L atreversing the effects of haloperidol was found to be 260 μg/kg (95%confidence interval: 180 to 370 μg/kg, R=100). This ED₅₀ value was notstatistically different (two-tailed t-test, t=0.67, df=6, P=0.53) fromthe ED₅₀ value (390 μg/kg) determined for NT69L-induced hypothermia at90 minutes. The ED₅₀ value for the blockade by NT69L of theapomorphine-induced climbing behavior was significantly lower than theED₅₀ values both for reversing haloperidol-induced catalepsy (t=2.74,df=15, P=0.0152) and hypothermia (t=3.88, df=17, P=0.0012).

Clozapine (a classical, atypical neuroleptic) was also tested for itsability to affect catalepsy caused by haloperidol. At a dosage of 20mg/kg (ip), clozapine did not cause catalepsy. When haloperidol (1mg/kg; ip) was injected 30 minutes before clozapine (20 mg/kg; ip), theanimals exhibited cataleptic effects similar to those observed inanimals receiving haloperidol alone. Pretreatment with clozapinefollowed by haloperidol, however, modulated the cataleptic effects ofhaloperidol. This modulation was not statistically significant Inaddition, the change in body temperature for the animals receivingtreatment with clozapine and/or haloperidol was in the range of −1° C.to −2° C. This temperature reduction range is markedly less than thatobserved in animal treated with NT69L alone.

These results indicate that NT69L, but not clozapine, completelyprevents catalepsy when given before haloperidol. These results alsoindicate that NT69L, but not clozapine, reverses haloperidol'scataleptic effects when given after haloperidol. Thus, NT69L may haveneuroleptic properties in humans and may be useful in the treatment ofextrapyramidal side effects caused by neuroleptics such as theirreversible tardive dyskinesia.

Example 8

Chronic Treatment with a Neo-Tryptophan-Containing Polypeptide

The effect of chronic injection of NT69L was tested. Rats were injectedwith NT69L (1 mg/kg; ip) daily, and tested for antinociception andhypothermia. Antinociception was measured using the hot plate testdescribed herein. Rats exhibited 100% MPE and about 4 degrees bodytemperature lowering after the first injection. After the secondinjection on day 2, however, no analgesic effect was observed and thebody temperature was lowered only 1.5 degrees. After the third injectionon day 3, there was still no analgesic effect and no body temperaturelowering was detected. In addition, the rats exhibited catalepsy wheninjected with haloperidol after the third and fourth days of NT69Linjection.

To check if the rats had developed tolerance to NT69L, the rats werechallenged with five times (5×) the dose of NT69L (5 mg/kg; ip) afterfour days of NT69L treatment at 1 mg/kg (ip). The rats exhibited areduction in body temperature comparable to the reduction normallyobserved in naive rats injected with NT69L (1 mg/kg; ip) for the firsttime. The haloperidol-induced cataleptic effect, however, was notreversed upon administration of the 5× dose of NT69L to the chronicallytreated rats (four day treatment with 1 mg/kg NT69L; ip). In addition,no analgesic effect was observed in the chronically treated rats afterchallenge with the 5× dose of NT69L.

The effect of chronic treatment with NT69L on the number of NT bindingsites within brain was assessed. Rats were treated daily with NT69L (1mg/kg; ip) for four days. On day five, the rats were treated with 5mg/kg (ip) NT69L, tested behaviorally, and then sacrificed. The PAG andrest of brain were dissected from the animal and used in NT bindinganalysis. Briefly, homogenates were prepared from freshly obtained PAGand the rest of brain of rats according to Goedert et al.(Brain Res.304, 71-81 (1984)) with the following modifications: the assay buffercontained the peptidase inhibitors 1,10 phenanthroline (1 μM) andaprotonin (5 mg/ml). For PAG and rest of brain binding assays, tissueswere incubated with 0.3 nM [¹²⁵I]NT (NEN, Boston, Mass.) at roomtemperature for 30 minutes. Total and nonspecific binding was measuredand the binding sites were normalized to polypeptide concentrations byBCA protein determination (Pierce Chemical Co., Rockford, Ill.).

Brain tissues from control rats exhibited more [¹²⁵I]NT binding thanbrain tissues from rats chronically treated with NT69L. Specifically,the PAG tissue from control rats contained 2.34 dpm/μg protein (n=2),while the same tissue from NT69L-treated rats contained 1.89 dpm/μgprotein (n=5). Likewise, the rest of brain tissue from control ratscontained 3.1 dpm/μg protein (n=2), while the same tissue fromNT69L-treated rats contained 2.1 dpm/μg protein (n=5). These resultsrepresent about a 20 to 30 percent reduction in [¹²⁵I]NT binding forbrain tissue from NT69L-treated animals.

Example 9

Weight Loss Properties of Neo-Tryptophan-Containing Polypeptides

Two groups of male Sprague-Dawley rats (small and large) and one groupof genetically obese Zucker rats were used to study the influence ofNT69L on various aspects of body weight. The group of smallSprague-Dawley rats weighed about 270 g at the beginning of the study,while the group of large Sprague-Dawley rats weighted about 400 g. Undernormal conditions, the small rats exhibit steady growth, and the largerats do not. All animals were individually housed in a room with a 12hour light/dark cycle. The rats had free access to commercial hard ratchow pellets and tap water. During the study, the rats received 100 μLof either saline only or 1 μg/kg, 10 μg/kg, or 1 mg/kg of NT69L on days1, 2, 7, 8, 11, and 12. Food intake (g), water consumption (mL), andbody weight (g) were recorded daily for 15 days. The results werepresented as mean±SEM, or as % of original weight. The data werecompared by variance analysis (unpaired or paired Student's t test) andRank sum test.

NT69L caused a significant (P<0.001) reduction in body weight gain wheninjected (ip) into small Sprague-Dawley rats at a dose of 1 μg/kg and 10μg/kg. The reduction in body weight gain was greatest one day afterinjection of NT69L. In addition, small rats failed to make-up for thereduction in body weight gain even after the NT69L administration wasdiscontinued. Specifically, small rats receiving saline only exhibited a29% increase in body weight by the end of 15 days, while small ratsreceiving 1 μg/kg NT69L exhibited only an 9.0% increase from theiroriginal body weight at day 15. Small rats receiving 10 μg/kg NT69Lexhibited an 8.4% increase from their original body weight at day 15.Food intake for the small rats was significantly (P<0.003) less than thefood intake observed for saline treated control animals throughout theexperiment, indicating that the observed reduction in body weight gainafter injection of NT69L is attributable in part to less food intake.

The large Sprague-Dawley rats injected (ip) with 1 mg/kg, but not with 1μg/kg or 10 μg/kg, exhibited a significant reduction in body weight(P<0.003). Specifically, the large rats receiving 1mg/kg NT69L exhibiteda 3.0% reduction in their original body weight, while saline treatedcontrol animals exhibited a 2.4% increase in their original body weightby day 15. In addition, food intake for the large rats was significantly(P<0.003) less than the food intake observed for saline treated controlanimals during the one to two days post NT69L injection. These resultsindicate that the observed reduction in body weight in NT69L-treatedanimals is attributable in part to less food intake.

The genetically obese Zucker rats injected (ip) with NT69L (1 mg/kg)also exhibited a significant reduction in weight gain (P<0.009).Specifically, NT69L-treated animals exhibited a 25% increase in theiroriginal body weight, while saline-treated control animals exhibited a31% increase in their original body weight by day 15. Again, food intakeby NT69L-treated animals was significantly reduced (P<0.01) during theone to two days post NT69L injection as compared to the food intake ofsaline-treated control animals. These results demonstrate the potenteffect of NT69L on (1) body weight gain reduction, (2) body weight loss,and (3) appetite when injected (ip) for two consecutive days at four tofive day intervals.

The effect of NT69L on blood hormone levels was assessed. Briefly,Sprague-Dawley rats were injected (ip) with either saline (n=20) or 1mg/kg NT69L (n=20). Five NT69L-treated rats and five control rats weresacrificed by decapitation at one, four, eight, and twenty-four hourspost-injection. Brains were harvested and dissected on ice. Thedifferent brain sections were kept on dry ice for HPLC analysis. Inaddition, blood was collected in cold centrifuge tubes with heparin andkept on ice. After collection, the blood was centrifuged at 2500 rpm forten minutes, and the plasma was collected and stored at −20° C. untilanalysis. Glucose, thyroxine (T4), thyroid stimulating hormone (TSH),and corticosterone levels were determined by enzyme assay or RIA.

Rats treated with NT69L (1 mg/kg; ip) exhibited a significant increasein blood glucose (P<0.005) and corticosterone (P<0.001) levels ascompared to the level observed in saline-treated controls. Specifically,NT69L-treated animals had a glucose level of 221 mg/dL and acorticosterone level of 24.6 μg/dL, while saline-treated animals had aglucose level of 130 mg/dL and a corticosterone level of 6.1 μg/dL atone hour post injection. In addition, rats treated with NT69L (1 mg/kg;ip) exhibited a significant reduction in TSH (P<0.001) and T4 (P<0.02)levels as compared to the level observed in saline-treated controls.Specifically, NT69L-treated animals had a TSH level of 0.9 mIU/L and aT4 level of 1.8 μg/dL, while saline-treated animals had a TSH level of7.65 mIU/L and a T4 level of 2.6 μg/dL at one hour post injection. By 24hours post-injection, the levels of blood glucose, corticosterone, TSH,and T4 had returned to the levels observed in control animals,indicating that the hyperglycemia as well as the inhibitory effect ofthyroid function due to NT69L were only transitory. The Zucker ratsexhibited a similar increase in blood glucose (310 mg/dL for theNT69L-treated vs. 140 mg/dL for the control) and corticosterone (19.7μg/dL for the NT69L-treated vs. 10.9 μg/dL for the control) levels, anda reduction in TSH (0.43 mIU/L for the NT69L-treated vs. 1.9 mIU/L forthe control) and T4 (1.03 μg/dL for the NT69L-treated vs. 1.47 μg/dL forthe control) levels one hour after injection (ip) of NT69L (1 mg/kg).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

30 1 13 PRT Artificial Sequence Synthetically generated peptide 1 XaaLeu Tyr Glu Asn Lys Pro Arg Arg Pro Tyr Ile Leu 1 5 10 2 6 PRTArtificial Sequence Synthetically generated peptide 2 Arg Arg Pro TyrIle Leu 1 5 3 5 PRT Artificial Sequence Synthetically generated peptide3 Arg Pro Tyr Ile Leu 1 5 4 6 PRT Artificial Sequence Syntheticallygenerated peptide 4 Arg Arg Pro Trp Ile Leu 1 5 5 6 PRT ArtificialSequence Synthetically generated peptide 5 Arg Arg Pro Tyr Xaa Leu 1 5 66 PRT Artificial Sequence Synthetically generated peptide 6 Xaa Lys ProTrp Xaa Leu 1 5 7 6 PRT Artificial Sequence Synthetically generatedpeptide 7 Xaa Arg Pro Tyr Ile Leu 1 5 8 6 PRT Artificial SequenceSynthetically generated peptide 8 Arg Xaa Pro Tyr Ile Leu 1 5 9 6 PRTArtificial Sequence Synthetically generated peptide 9 Arg Arg Pro XaaIle Leu 1 5 10 6 PRT Artificial Sequence Synthetically generated peptide10 Arg Arg Pro Xaa Ile Leu 1 5 11 6 PRT Artificial SequenceSynthetically generated peptide 11 Arg Arg Pro Xaa Ile Leu 1 5 12 6 PRTArtificial Sequence Synthetically generated peptide 12 Arg Arg Pro XaaXaa Leu 1 5 13 6 PRT Artificial Sequence Synthetically generated peptide13 Xaa Arg Pro Xaa Xaa Leu 1 5 14 6 PRT Artificial SequenceSynthetically generated peptide 14 Xaa Arg Pro Xaa Xaa Leu 1 5 15 6 PRTArtificial Sequence Synthetically generated peptide 15 Xaa Arg Pro XaaIle Leu 1 5 16 6 PRT Artificial Sequence Synthetically generated peptide16 Xaa Lys Pro Xaa Xaa Leu 1 5 17 6 PRT Artificial SequenceSynthetically generated peptide 17 Xaa Arg Pro Xaa Xaa Leu 1 5 18 6 PRTArtificial Sequence Synthetically generated peptide 18 Xaa Xaa Pro XaaXaa Leu 1 5 19 5 PRT Artificial Sequence Synthetically generated peptide19 Xaa Pro Xaa Xaa Leu 1 5 20 5 PRT Artificial Sequence Syntheticallygenerated peptide 20 Xaa Pro Xaa Ile Leu 1 5 21 5 PRT ArtificialSequence Synthetically generated peptide 21 Xaa Pro Xaa Xaa Leu 1 5 22 5PRT Artificial Sequence Synthetically generated peptide 22 Xaa Pro XaaIle Leu 1 5 23 6 PRT Artificial Sequence Synthetically generated peptide23 Arg Xaa Pro Xaa Ile Leu 1 5 24 6 PRT Artificial SequenceSynthetically generated peptide 24 Arg Xaa Pro Xaa Xaa Leu 1 5 25 8 PRTArtificial Sequence Synthetically generated peptide 25 Asp Arg Val TyrIle His Pro Phe 1 5 26 8 PRT Artificial Sequence Synthetically generatedpeptide 26 Asp Arg Val Xaa Ile His Pro Phe 1 5 27 9 PRT ArtificialSequence Synthetically generated peptide 27 Arg Pro Pro Gly Phe Ser ProPhe Arg 1 5 28 9 PRT Artificial Sequence Synthetically generated peptide28 Arg Pro Pro Gly Xaa Ser Pro Phe Arg 1 5 29 5 PRT Artificial SequenceSynthetically generated peptide 29 Tyr Gly Gly Phe Leu 1 5 30 5 PRTArtificial Sequence Synthetically generated peptide 30 Xaa Gly Gly PheLeu 1 5

What is claimed is:
 1. A method of synthesizing neo-tryptophan, saidmethod comprising: a) providing 4-hydroxymethyl indole, and b)substituting the hydroxyl group of said 4-hydroxymethyl indole with aglycyl unit to produce neo-tryptophan.
 2. The method of claim 1, whereinthe N-1 nitrogen of said 4-hydroxymethyl indole is protected by aprotecting group, said method comprising removing said protecting groupafter said substitution.